QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mesa, R. Articles by Mayorga, L. S. Search for Related Content PubMed PubMed Citation Articles by Mesa, R. Articles by Mayorga, L. S.

Rab22a affects the morphology and function of the endocytic pathway

¹ Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina
² Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA

View larger version (101K): [in a new window]   Fig. 1. GFP-Rab22a localizes to early and late endosomes. CHO cells were transfected with pEGFP-Rab22aWT and 24 hours later the distribution of the GFP fluorescence was recorded. Early endosomes were labeled by a 5 minute uptake of Rh-HRP (A-C) or Rh-Tf (D-F). Late endosomes were labeled by a 5 minute uptake of Rh-HRP followed by 15 minute chase (G-I) or by immunostaining with an anti CI-M6PR antibody (J-L). Notice the large size of some Rab22a-positive vacuoles (arrow heads) and the altered morphology of early and late compartments in cells overexpressing Rab22a (t) when compared with untransfected cells (u). The inset in B shows tubular projections attached to a large vesicle. The bars represent 7 µm.

View larger version (90K): [in a new window]   Fig. 2. GFP-Rab22a does not localize with Golgi structures or lysosomes. CHO cells were transfected with pEGFP-Rab22aWT and 24 hours later the distribution of the GFP fluorescence was analyzed. The Golgi apparatus was labeled with an antibody that recognizes membrin, a cis Golgi marker (A-C). Lysosomes were loaded with Rh-HRP by a 1 hour uptake followed by 1 hour chase (D-F). Acidic compartments were labeled with LysoTracker red (G-I). Autophagosomes were labeled with MDC (monodansylcadaverine) (J-L). Very scarce colocalization can be observed with the four markers used. The bars represent 7 µm.

View larger version (62K): [in a new window]   Fig. 3. Rab22aS19N, a mutant with low affinity for GTP, is mostly cytosolic, whereas Rab22aQ64L, a mutant with low GTPase activity, localizes in large vesicles that are labeled by early and late endosomal markers. Cells expressing GFP-Rab22aS19N presented a diffuse cytosolic fluorescence. The protein was enriched in a perinuclear region that colocalized with a Golgi marker (BODIPY-TR ceramide) (A-C). The distribution of the Rab22aQ64L mutant was completely different. This GTPase-deficient mutant localized to large ring-shaped vacuoles that were labeled by a 5 minute uptake of Rh-HRP (D-F) or Rh-Tf (G-I). The protein also localized to late endosomes labeled by a 5 minute uptake of Rh-HRP followed by a 15 minute chase (J-L) or by immunocytochemistry using an anti CI-M6PR antibody (M-O). In some cells, Rab22aQ64L was also present at the plasma membrane (arrow). The bars represent 7 µm.

View larger version (64K): [in a new window]   Fig. 4. Rab22aQ64L, the GTPase-deficient mutant, colocalizes not only with early and late endosomes, but also with lysosomes and autophagosomes. Lysosomes were loaded with Rh-HRP by a 1 hour uptake followed by a 1 hour chase (A-C). Acidic compartments were labeled with LysoTracker red (D-F). Autophagosomes were labeled with MDC (monodansylcadaverine) (G-I). Enlarged lysosomal and autophagosomal compartments were evident in cells overexpressing Rab22aQ64L (t) when compared with untransfected cells (u). The bars represent 7 µm.

View larger version (31K): [in a new window]   Fig. 5. Overexpression of Rab22a affects endocytosis. (A) CHO cells transiently expressing GFP alone (vector) or GFP fused to Rab22aWT, Rab22aS19N and Rab22aQ64L were allowed to endocytose HRP for 1 hour. The cells were washed and the amount of peroxidase associated with the cells was measured. The uptake was expressed as a percentage of the HRP endocytosed by untransfected cells (control). (B) The same experiment was performed with CHO clones stably expressing Rab22a wild-type and the mutants. (C) CHO cells that had internalized 125I-HRP as in A were washed and incubated for 40 minutes. The amount of HRP digested (dark gray bars) and recycled into the medium (light gray bars) was estimated from trichloroacetic acid soluble and precipitable radioactivity as explained under Material and Methods. The data are expressed as a percentage of HRP present in the cells after the internalization period. Black bars show the percentage of HRP that is lost from the cells by digestion and recycling. The data represent the mean±s.e.m. of five experiments. Asterisks indicate significant differences from the control (P<0.05).